Christian Doppler Laboratory for Innovative Tools for Biosimilar Characterization, University of Salzburg, Hellbrunner Straße 34, 5020, Salzburg, Austria.
Department of Biosciences, University of Salzburg, Hellbrunner Straße 34, 5020, Salzburg, Austria.
Sci Rep. 2019 Feb 1;9(1):1111. doi: 10.1038/s41598-018-37624-1.
Detailed analysis of biopharmaceuticals is crucial for safety, efficacy and stability. Aptamers, which are folded, single-stranded oligonucleotides, can be used as surrogate antibodies to detect subtle conformational changes. We aimed to generate and assess DNA aptamers against the therapeutic anti-CD20 antibody rituximab. Six rituximab-specific aptamers with K = 354-887 nM were obtained using the magnetic bead-based systematic evolution of ligands by exponential enrichment (SELEX) technology. Aptamer folds were analysed by online prediction tools and circular dichroism spectroscopy suggesting quadruplex structures for two aptamers while others present B-DNA helices. Aptamer binding and robustness with respect to minor differences in buffer composition or aptamer folding were verified in the enzyme-linked apta-sorbent assay. Five aptamers showed exclusive specificity to the Fab-fragment of rituximab while one aptamer revealed a broader recognition pattern to other monoclonal antibodies. Structural differences upon incubation at 40 °C for 72 h or UV exposure of rituximab were uncovered by four aptamers. High similarity between rituximab originator and biosimilar lots was demonstrated. The most sensitive aptamer (RA2) detected signal changes for all lots of a copy product suggesting conformational differences. For the first time, a panel of rituximab-specific aptamers was generated allowing the assessment of conformational coherence during production, storage, and biosimilarity of different products.
对生物制药进行详细分析对于安全性、疗效和稳定性至关重要。适体是折叠的单链寡核苷酸，可作为替代抗体来检测细微的构象变化。我们旨在生成和评估针对治疗性抗 CD20 抗体利妥昔单抗的 DNA 适体。使用基于磁珠的指数富集配体系统进化（SELEX）技术获得了 6 种对利妥昔单抗具有特异性的适体，其 Kd 值为 354-887 nM。通过在线预测工具和圆二色性光谱分析，两种适体的折叠结构被分析为四联体，而其他适体呈现 B-DNA 螺旋。在酶联适体吸附测定中，验证了适体结合和对缓冲组成或适体折叠的微小差异的稳健性。5 种适体对利妥昔单抗的 Fab 片段表现出特异性，而 1 种适体对其他单克隆抗体表现出更广泛的识别模式。通过 4 种适体揭示了在 40°C 孵育 72 小时或利妥昔单抗暴露于 UV 下时的结构差异。显示了利妥昔单抗原研药和生物类似药批次之间的高度相似性。最敏感的适体（RA2）检测到所有批次的拷贝产品的信号变化，提示存在构象差异。首次生成了一组利妥昔单抗特异性适体，可用于评估生产过程、储存过程中和不同产品的生物类似性期间的构象一致性。
